The principal goal of this project is to understand the impact of HIV envelope-mediated signaling on viral replication and immune dysfunction. This information may facilitate the discovery of new strategies for the treatment of HIV infection. HIV envelope proteins, which are displayed on the surface of viral particles, bind to two receptors on T-lymphocytes in order to facilitate infection of those cells. Initially envelope proteins on the outer surface of virions dock with the CD4 receptor, and subsequently engage a co-receptor, either CCR5 or CXCR4. Envelope engagement of each of these receptors results in signal transduction; however the consequences of this signaling is only partially understood. In previous studies we demonstrated that HIV envelope-mediated signaling induces several biological responses in primary T-cells and macrophages, including the induction of proinflammatory cytokines and increased rate of apoptosis. More recently it has been shown that envelope also binds to DC-SIGN, a C-type lectin receptor. We have determined that gp120 binding to DC-SIGN on dendritic cells also mediates biological responses. We evaluated the response of monocyte derived dendritic cells (MDDCs) to a panel of recombinant oligomeric envelope proteins with different glycosylation content. Modulation of gene expression was evaluated using oligonucleotide microarrays, and protein phosphorylation events were evaluated with high throughput proteomic analysis. MDDC responses were strongly influenced by the carbohydrate content of gp120. We identified genes upregulated only by a hyperglycosylated gp120 and genes upregulated only by a hypoglycosylated gp120. We are also evaluating gp120 interactions with other C-type lectin receptors that are expressed on other cell types. [unreadable] Understanding how various cell types respond to gp120 may provide further insight into the basis of HIV induced immune dysfunction and pathogenesis. Of particular interest is the interaction of gp120 with plasmacytoid dendritic cells (pDCs). pDCs, which play a central role in innate and adaptive immune responses against viral infections, secrete type I IFNs and proinflammatory cytokines upon stimulation via either TLR7 or TLR9. Throughout the course of HIV infection, the production of type-I IFNs is profoundly impaired, and total pDC cell counts in peripheral blood correlates inversely with viral load and positively with CD4+ T-cell count. The origin of these defects is unclear. pDCs express CD4, CCR5 and CXCR4, the primary receptor and co-receptors, respectively, for the HIV envelope, yet little is known about the effects of the viral envelope on these cells. We determined that exposure of pDCs to gp120 results in the suppression of activation of these cells. This suppression was specific for TLR9-mediated responses, since TLR7-mediated responses were unaffected by gp120. gp120 also suppressed TLR9-mediated induction of proinflammatory cytokines and the expression of CD83, a marker of DC activation. Finally, gp120 suppressed pDC-induced cytolytic activity of NK cells. Taken together, these data demonstrate that the direct interaction of HIV-1 gp120 with pDCs interferes with TLR9 activation resulting in a decreased ability of pDCs to secrete antiviral and inflammatory factors that typically play a central role in initiating host immune responses against invading pathogens.